Difference between revisions of "Part:BBa K2074025"
 
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<partinfo>BBa_K2074025 short</partinfo>
 
<partinfo>BBa_K2074025 short</partinfo>
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There is a overview about parts which 2016 FAFU-CHINA created.
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https://static.igem.org/mediawiki/igem.org/thumb/9/9f/T--FAFU-CHINA--partsclones.png/800px-T--FAFU-CHINA--partsclones.png<br />
 
https://static.igem.org/mediawiki/igem.org/thumb/9/9f/T--FAFU-CHINA--partsclones.png/800px-T--FAFU-CHINA--partsclones.png<br />
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our part is cyt2 which have toxicity to the larve of mosquit .We transferred the gene cyt2 into plasmid ,and transformed the recombinant plasmid into Chlamydomonas reinhardti to express protein .
 
  
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Cyt2 is cloned from Bacillus thuringiensis BRC-LLP29. It shows specific toxicity to Culex and Aedes by bioassay.Cyt proteins have a single α-β domain which do not bind to receptors but can directly insert into the cell membrane and then form a pore causing cell death. Although Cry and Cyt proteins are two big families of δ-endotoxins, they are far related.Cyt1 and Cyt2 are two types of Cyt proteins found in Bti.. Generally, Cry proteins are believed to exert toxicity by interacting with the proteins on the brush border membrane and then insert into the membrane which takes multiple steps. At the beginning in mosquitoes' gut, the crystalline inclusions are cleaved at the disulfide bond to release the Cry pre-toxin. Then the soluble proteins are activated by being cleaved again by intestinal protease. When toxins reach to the brush border membrane microvilli, they bind to the proteins, or known as receptors on the membrane. The binding process takes two step. Firstly, the monomeric Cry toxin binds to cadherin, resulting in the formation of pre-pore oligomer as Figure1.
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[[File:FAFU-CHINA 2016 P1.png|600px|thumb|center|'''Figure.1''' The Mechanism of Cry and Cyt Toxins]]
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Then the oligomer binds to a GPI-anchored APN or ALP. Secondly, the previous binding induces the oligomer insertion into the lipid rafts membrane. A formation of ion permeable pore is followed by the insertion which allows small molecules to pass through the membrane. The membrane potential inevitably changes greatly, causing the swelling of cell and finally breaking down. When the cell lysis reaches to a certain degree, the midgut necrosis and epithelial denaturation follow. Then, the alkaline hypertonic inclusions in midgut enters into hemocoel and the pH of haemolymph rises causing paralysis of larvae and finally death.
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To increase the express level in Chlamydomonas reintmrdtii, we synthesis Chlamydomonas reintmrdtii codon optimized Cyt2. Meanwhile, we added 2A peptide sequence at the end of 5’ According to the published paper and the result of Swiss-model, there is no effect to the toxicity of Cry or Cyt. Users can use infusion technology to link the express vector to express toxins.
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[[File:T--FAFU-CHINA--BBa_K2074025.png|600px|thumb|center]]
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 13:27, 23 October 2016


Cyt2(Codon optimization)-Extended FMDV

There is a overview about parts which 2016 FAFU-CHINA created.

800px-T--FAFU-CHINA--partsclones.png

Cyt2 is cloned from Bacillus thuringiensis BRC-LLP29. It shows specific toxicity to Culex and Aedes by bioassay.Cyt proteins have a single α-β domain which do not bind to receptors but can directly insert into the cell membrane and then form a pore causing cell death. Although Cry and Cyt proteins are two big families of δ-endotoxins, they are far related.Cyt1 and Cyt2 are two types of Cyt proteins found in Bti.. Generally, Cry proteins are believed to exert toxicity by interacting with the proteins on the brush border membrane and then insert into the membrane which takes multiple steps. At the beginning in mosquitoes' gut, the crystalline inclusions are cleaved at the disulfide bond to release the Cry pre-toxin. Then the soluble proteins are activated by being cleaved again by intestinal protease. When toxins reach to the brush border membrane microvilli, they bind to the proteins, or known as receptors on the membrane. The binding process takes two step. Firstly, the monomeric Cry toxin binds to cadherin, resulting in the formation of pre-pore oligomer as Figure1.

Figure.1 The Mechanism of Cry and Cyt Toxins

Then the oligomer binds to a GPI-anchored APN or ALP. Secondly, the previous binding induces the oligomer insertion into the lipid rafts membrane. A formation of ion permeable pore is followed by the insertion which allows small molecules to pass through the membrane. The membrane potential inevitably changes greatly, causing the swelling of cell and finally breaking down. When the cell lysis reaches to a certain degree, the midgut necrosis and epithelial denaturation follow. Then, the alkaline hypertonic inclusions in midgut enters into hemocoel and the pH of haemolymph rises causing paralysis of larvae and finally death. To increase the express level in Chlamydomonas reintmrdtii, we synthesis Chlamydomonas reintmrdtii codon optimized Cyt2. Meanwhile, we added 2A peptide sequence at the end of 5’ According to the published paper and the result of Swiss-model, there is no effect to the toxicity of Cry or Cyt. Users can use infusion technology to link the express vector to express toxins.

T--FAFU-CHINA--BBa K2074025.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 137
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 271
  • 1000
    COMPATIBLE WITH RFC[1000]